LHRH antagonists

ABSTRACT

Disclosed herein are analogues of the luteinizing hormone-releasing hormone (LH-RH), which are potent antagonists of LH-RH. These peptides inhibit the release of gonadotropins from the pituitary in mammals, including humans and possess antitumor activity. 
     Formula I represents peptides which are within the scope of this invention: 
     
         X--R.sup.1 --R.sup.2 --R.sup.3 --Ser--R.sup.5 --R.sup.6 
    
      (AY 2 )--Leu--Arg--Pro--D--Ala--NH 2                I 
     and the pharmaceutically acceptable salts thereof, wherein 
     R 1  is D-Phe, D-Phe(4Cl), D-Nal(1) or D-Nal(2), 
     R 2  is D-Phe or D-Phe(4HI), 
     R 3  is D-Trp, D-Phe, D-Phe(4HI), D-Nal(1), D-Nal(2) or D-Pal(3), 
     R 5  is Tyr or Arg, 
     R 6  is D-Lys or D-Orn, 
     HI is fluoro, chloro or bromo 
     X is a lower alkanoyl group of 2-5 carbon atoms, 
     A is a diaminoacyl residue having the formula ##STR1##  where m is 0 or 1, 
     n is 0 or 1, 
     Y is Y 1  or Y 2 , wherein 
     Y 1  is an acyl group derived from straight or branched chain aliphatic, alicyclic carboxylic acids having from 3 to 12 carbon atoms or aromatic carboxylic acid of 6 or 10 ring carbon atoms, 
     Y 2  is carbamoyl or alkyl-substituted carbamoyl group having the formula 
     
         H--(CH.sub.2).sub.n --NH--CO--                             III 
    
      where n is 0-3.

This invention was made with Government support under grant Nos. 40003and 40004, awarded by the N.C.I. (NIH). The U.S. Government has certainrights in this application.

RELATED APPLICATIONS

This application is a continuation-in-part of copending application,Ser. No. 07/404,667 filed Sep. 7, 1989, which is a continuation-in-partof application Ser. No. 260,994 filed Oct. 21, 1988, now abandoned.

BACKGROUND OF THE INVENTION

The present invention is directed to novel peptides having an inhibitoryeffect on the release of gonadotropins by the pituitary in mammals,including humans and having an influence on the growth of canceroustumors in humans. More specifically, the present invention relates toantagonistic analogs of luteinizing hormone-releasing hormone (LHRH),which have the structure:

    pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2

salts thereof, and to pharmaceutical compositions and methods of usepertaining to these analogs.

DISCUSSION OF THE PRIOR ART

Hypothalamic luteinizing hormone-releasing hormone (LHRH) controlspituitary synthesis and secretion of gonadotropins (LH and FSH) that areessential for the regulation of the synthesis of sex steroids in thegonads.

Over 2500 new, synthetic analogs of LHRH (agonistic and antagonisticanalogs) have been reported since its discovery and structuralelucidation (A. V. Schally et al., Fertil. Steril. 22, 703-721, 1971) inview of their expected medical applications (M. J. Karten and J. E.Rivier, Endocrine Rev. 7, 44-66, 1986; A. Dutta, Drugs of the Future, 13761-787, 1988). LHRH antagonists compete with endogeneous LHRH at thehypophysial receptors and directly inhibit the secretion ofgonadotropins. They have significant therapeutic advantages over theagonists in that they almost immediately inhibit gonadotropin secretionwithout inducing an initial rise in gonadotropins, as is characteristicof LHRH agonists. Antagonists of LHRH have been used in endocrinologyand gynecology to control fertility and treatment of precocious puberty,as they block ovulation in the female and suppress spermatogenesis inthe male. The use of antagonists in oncology for treatment ofhormone-sensitive tumors is very recent, but most promising (A. V.Schally et al., in: GnRH analogs in cancer and in human reproduction.Basic Aspects, (edited by B. H. Vickery and V. Lunenfeld), KluwerAcademic Publishers, Dordrecht/Boston/London, Vol. 1, pp. 5-31, 1989).

The most interesting antagonists to date have been compounds whosestructure is a modification of the structure of LHRH. Systematicmodification of the molecule showed the contribution of the individualamino acids and their side chains to the biological activity. Theearlier most potent antagonists frequently had a cluster of hydrophobicD-amino acid residues at the N-terminal and strongly basic, hydrophilicD-amino acids at position 6 and/or 8 (D. H. Coy et al., Endocrinology,100 1445-1447, 1982; A. Horvath et al., Peptides 3 969-971, 1982; J.Rivier et al., J. Med. Chem. 29, 1846-1851, 1986). However, thesepotent, hydrophilic antagonists caused transient systemic edema of theface and extremities and inflamation at the injection site when injectedsubcutaneously into rats at 1.25 or 1.5 mg/kg body weight. Theseanalogues, which are mast cell secretagogues, release histamine and,when given intravenously to rats at a dose of 1.25 mg/kg body weight,can also cause cyanosis and respiratory depression leading to cell death(Smith et al., Contraception 29, 283-289, 1984; Morgan et al., Int.Arch. Allergy Appl. Immunology 80, 70-75, 1986). To overcome these sideeffects but maintain the high antiovulatory potency of the antagonists,research was directed towards the change of the basicity of the sidechains at the region of 5-8 amino acids. Hocart et al. (J. Med. Chem.30, 1910-1914, 1987) found that the substitution of alkylated Lysderivatives in position 6 did not produced any significant changes inthe histamine releasing activity of the analogues whereas similarsubstituents at position 8 reduced the histamine release 10-fold.Detirelix [Ac-D-Nal(2)¹,D-Phe(4Cl)²,D-Trp³,D-hArg(Et)₂ ⁶,D-Ala¹⁰ ]proved to be a powerful antagonist (L. A. Adams et al., J. Clin.Endocrinol. Metab. 62, 58, 1986) but has hypotensive and bradycardicside effect (C. H. Lee et al., Life Sci., 45, 67, 1989). Antagonistsnamed Nal-Glu-GnRHant retain ovulation inhibition potency and havemarkedly less in vitro histamine-releasing activity (J. E. Rivier etal., J. Med. Chem. 29, 1846-1851, 1986), but local allergic response insome human subjects remains a concern. Introduction of N.sup.ε-nicotinoyl-lysine into positions 5,6 and N.sup.ε -isopropyllysine intoposition 8 led to a compound with high antiovulatory and negligiblehistamine releasing activity (Ljungqvist et al., Proc. Natl. Acad. Sci.USA, 85 8236-8240, 1988). The modification by Bajusz et al. (Int. J.Pept. Prot. Res., 32 425-435, 1988) i.e. incorporation of citrulline andhomocitrulline into position 6 produced peptides having no edematogenicand anaphylactoid side effects and high inhibitory effect, asexemplified byAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH₂.Some of these compounds were found to have inhibiting effect on growthof various animal tumor models in vivo and to suppress growth ofdifferent human cancer cell lines (A. V. Schally, in General Gynecology,Vol. 6., Parthenon Press, Carnforth, England, 1989, pp. 1-20; Szende etal., J. Natl. Cancer Inst., 82, 513-517, 1990; Szende et al., CancerResearch), 50, 3716-3721, 1990; E. Korkut et al., Proc. Natl. Acad. Sci.US, accepted for publication) and thus might be potential therapeuticagents in the treatment of different cancers (prostate, breast,endometrial, ovarian and pancreatic).

Many human tumors are hormone dependent or hormone-responsive andcontain hormone receptors; e.g., mammary carcinomas contain estrogen,progesterone, glucocorticoid, LHRH, EGF, IGF-I. and somatostatinreceptors. Peptide hormone receptors have also been detected in acuteleukaemia, prostate-, breast-, pancreatic, ovarian-, endometrial cancer,colon cancer and brain tumors (M. N. Pollak, et al., Cancer Lett. 38223-230, 1987; F. Pekonen, et al., Cancer Res., 48 1343-1347, 1988; M.Fekete, et al., J. Clin. Lab. Anal. 3 137-147, 1989; G. Emons, et al.,Eur. J. Cancer Oncol., 25 215-221, 1989). Our recent findings (M.Fekete, et al., Endocrinology, 124 946-955, 1989; M. Fekete, et al.Pancreas 4 521-528, 1989) have revealed that both agonistic andantagonistic analogs of LHRH bind to human breast cancer cell membranes,and also to the cell membranes of pancreatic cancer although the lattertumor thought to be hormone-independent. It has been demonstrated thatbiologically active peptides such as melanotropin (MSH), epidermalgrowth factor, insulin and agonistic and antagonistic analogs of LHRH(L. Jennes, et. al., Peptides 5 215-220, 1984) are internalized by theirtarget cells by endocytosis.

SUMMARY OF THE INVENTION

The present invention refers to novel antagonistic decapeptide analoguesof hypothalamic LHRH which possess high antiovulatory and antineoplasticactivity, and are free of anaphylactoid side effects and are believed tobe free of endematogenic effects.

The compounds of this invention are represented by Formula I

    X--R.sup.1 --R.sup.2 --R.sup.3 -Ser-R.sup.5 --R.sup.6 (AY.sub.2)-Leu-Arg-Pro-D-Ala-NH.sub.2                     I

wherein

R¹ is D-Phe, D-Phe(4Cl), D-Nal(1) or D-Nal(2),

R² is D-Phe or D-Phe(4Hl),

R³ is D-Trp, D-Phe, D-Phe(4Hl), D-Nal(1), D-Nal(2) or D-Pal(3),

R⁵ is Tyr or Arg,

R⁶ is D-Lys or D-Orn,

Hl is fluoro, chloro or bromo

X is a lower alkanoyl group of 2-5 carbon atoms,

A is a diaminoacyl residue having the formula ##STR2## where m is 0 or1,

n is 0 or 1,

Y is Y¹ or Y², wherein

Y¹ is an acyl group derived from straight or branched chain aliphatic oralicyclic carboxylic acids having from 3 to 12 carbon atoms or aromaticcarboxylic acid of 6 or 10 ring carbon atoms,

Y² is a carbamoyl group or C₁ -C₅ alkyl carbamoyl group having theformula

    H--(CH.sub.2).sub.n --NH--CO--                             III

where n is 0-3.

The therapeutically acceptable salts of the compound of Formula I areincluded within the scope of this invention.

The peptides of Formula I can be synthesized by classical solutionpeptide synthesis or preferably, solid phase technique usingmethylbenzylhydrylamine (MBHA), benzhydrylamine (BAH) resin or2-methoxy-4-alkoxybenzyl alcohol (Sasrin) resin with a suitable amidolinker.

Such method provides intermediate peptides and/or intermediatepeptide-resins of Formula IV:

    X.sup.1 --R.sup.1 --R.sup.2 --R.sup.3 --Ser(X.sup.4)--R.sup.5 (X.sup.5)--R.sup.6 (X.sup.6)-Leu-Arg(X.sup.8)-Pro-D-Ala-NH--X.sup.10IV

wherein

R¹, R², R³, R⁵, and R⁶ are as defined above,

X¹ is a lower alkanoyl group of 2-5 carbon atoms,

X⁴ is hydrogen or a protecting group for the Ser hydroxyl group,

X⁵ is hydrogen or a protecting group for the Tyr phenolic hydroxylgroup, or a protecting group for the guanidino group of Arg,

X⁶ is hydrogen or a protecting group for the Lys, Orn,

X⁸ is hydrogen or a protecting group for the Arg guanidino group,

X¹⁰ is hydrogen or linking (spacer) group incorporated into a resin.

To insure the selective reactions on the diamino-alkanoyl side chain ofR⁶ to get peptides of Formula I, intermediate peptides of Formula V areprepared by solid phase method as peptides of Formula I with theexception that suitably protected R⁶ [A(X^(6'))₂ ] is incorporated inplace of R⁶ (X⁶) in position 6:

    X.sup.1 --R.sup.1 --R.sup.2 --R.sup.3 --Ser(X.sup.4)--R.sup.5 (X.sup.5)--R.sup.6 [A(X.sup.6').sub.2 ]-Leu-Arg(X.sup.8)-Pro-D-Ala-NH--X.sup.10                 V

wherein

X¹, R¹, R², R³, R⁵, R⁶, A are as defined above, X¹, X⁴, X⁵ and X⁸ are asdefined above but not hydrogen,

X^(6') is hydrogen or a protecting group of the diamino side chain,

X¹⁰ is a linkage group incorporated into a resin.

To prepare compounds of Formula I wherein Q is A(Y¹)₂ or A(Y²)₂ fourdifferent reaction schemes have been utilized:

a) Intermediate peptides of Formula IV (wherein R¹, R², R³, R⁵, R⁶ andX¹ are as defined above, X⁴, X⁵, X⁶, X⁸ and X¹⁰ are hydrogen) arereacted with preformed A(Y¹)₂ or A(Y²)₂, wherein A, Y¹ and Y² aredefined as above.

b) Alternatively, compounds of Formula V (wherein R¹, R², R³, R⁵, R⁶ andX¹ are as defined above, X⁴, X⁵ are side chain protecting groups, X^(6')is hydrogen and X¹⁰ is linkage group of the resin) are used asintermediate peptides. Compounds of Formula I, wherein Y is Y¹ producedby direct acylation of intermediate peptide of Formula V with anacyl-halide or -anhydride, followed by splitting the peptides from theresin and removing the protecting groups in one step.

c) According to another method, R⁶ [A(Y₂)] is prepared in advance byreacting a suitable protected R⁶ with A then with Y, or with preformedA(Y)₂ and followed by incorporation into the peptide during the solidphase peptide synthesis.

d) The two free amino groups of A of intermediate peptides of Formula V(wherein R¹, R², R³, R⁵, R⁶, A and X¹ are as defined above, X⁴, X⁵,X^(6'), X⁸ and X¹⁰ are hydrogen) are acylated with acyl-imidazole.

The invention also provides methods for splitting off one or moreprotecting group(s) and/or cleaving the peptides from the resin support,for purifying a synthesized peptide and converting it into a nontoxic,pharmaceutically acceptable salt, wherein the salts retain the desiredbiological activity of the parent compound.

The peptides of this invention inhibit the ovulation of female rats atdosages of less 0.15-1.0 μg/kg body weight, when administered s.c. atabout noon on the day of proestrus. These peptides have a long actingeffect in suppressing the LH, FSH and testosterone levels when they areinjected into castrated male rats at doses of 0.5-2.0 micrograms/kg bodyweight. Peptides 7 and 8 induced significant decrease in the LH levelsfor more than 24 hours (p<0.01). Forty-eight hours after injection, bothantagonists showed significant inhibition (p<0.05) at a dose of 5 μg. Atthat time, Peptide 8 was active even at a dose of 1.25 μg (p<0.05). Themajority of the compounds of Formula I show high affinity for membranereceptors of rat pituitaries and humane breast cancers. In cytotoxicitytest, in cultures of human breast and prostate cancer cell lines, someanalogues powerfully inhibit the ³ H-thymidine incorporation.

The inhibition of growth of Dunning R3327H prostate cancer has beendemonstrated after treatment of rats with Peptide 8. Tumor doubling timewas increased to 42 days comparing to the 12 days of the control group.The body weight did not change during the treatment, however the weightof testis, seminal vesicles and ventral prostate were greatly reduced inthe group which received Peptide 8. The results indicated that Peptide8, released from sustained delivery systems can effectively suppress thegrowth of prostate cancers.

A pharmaceutical composition is provided by admixing the compound ofFormula I with a pharmaceutically acceptable carrier includingmicrocapsules (microspheres) or microgranules (microparticles)formulated from poly(DL-lactide-co-glycolide) for sustained delivery.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For convenience in describing this invention, the conventionalabbreviations for the amino acids, peptides and their derivatives areused as generally accepted in the peptide art and as recommended by theIUPAC-IUB Commission on Biochemical Nomenclature [European. J. Biochem.,138, 9-37 (1984)].

The abbreviations for the individual amino acid residues are based onthe trivial name of the amino acid, e.g. pGlu is pyroglutamic acid, Hisis histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Lys islysine, Orn is ornithine, Leu is leucine, Arg is arginine, Pro isproline, Gly is glycine, Ala is alanine and Phe is phenylalanine. Wherethe amino acid residue has isometric forms, it is the L-form of theamino acid that is represented unless otherwise indicated.

Abbreviations of the uncommon amino acids employed in the presentinvention are as follows: A₂ pr is 2,3-diaminopropionic acid, A₂ bu is2,4-diaminobutyric acid, Nal(2) is 3-(2-naphthyl)alanine, D-Pal(3) is3-(3-pyridyl)alanine, Phe(4Cl) is 4-chlorophenylalanine.

Peptide sequences are written according to the convention whereby theN-terminal amino acid is on the left and the C-terminal amino acid is onthe right.

Other abbreviations used are:

AcOH acetic acid

Ac₂ O acetic anhydride

Boc tert.butoxycarbonyl

Bz benzoyl

Bzl benzyl

Car Carbamoyl

CHC Cyclohexanoyl

DCB 2,6-dichlorobenzyl

DCC N,N'-dicyclohexylcarbodiimide

DCM dichloromethane

DIC N,N'-diisopropylcarbodiimide

DMF dimethylformamide

EtCar Ethyl Carbamoyl

FMOC Fluorenylmethyloxycarbonyl

HOBt 1-hydroxybenzotriazole

HOPCP pentachlorophenol

HPLC high-performance liquid-chromatography

iPrOH iso-propylalcohol

LAU lauryl

MeCN acetonitrile

MeOH methyl alcohol

OSu N-hydroxy succinamide ester

PRL propionyl

TEA triethylamine

TFA trifluoroacetic acid

Tos 4-toluenesulfonyl

Z(2-Cl) 2-chloro-benzyloxycarbonyl

Z benzyloxycarbonyl

Compounds which are especially preferred embodiments of the presentinvention have the structure:

    X--R.sup.1 --R.sup.2 --R.sup.3 -Ser-R.sup.5 --R.sup.6 (AY.sub.2)-Leu-Arg-Pro-D-Ala-NH.sub.2                     I

wherein,

R¹ is D-Nal(2);

R² is D-Phe(4Cl),

R³ is D-Trp or D-Pal(3),

R⁵ is Tyr or Arg,

R⁶ is D-Lys or D-Orn,

X is acetyl.

A is A₂ pr or DL-A₂ bu,

Y is Y¹ or Y²,

wherein

Y¹ is formyl, acetyl, propionyl, butyryl, i-butyryl, cyclohexanoyl orbenzoyl,

Y² is carbamoyl, N-methyl-carbamoyl or N-ethyl-carbamoyl.

The most particularly preferred embodiments are:

1. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(Car)₂]-Leu-Arg-Pro-D-Ala-NH₂,

2. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂,

3. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(For)₂]-Leu-Arg-Pro-D-Ala-NH₂,

4. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(EtCar)₂]-Leu-Arg-Pro-D-Ala-NH₂,

5. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(CHC)₂]-Leu-Arg-Pro-D-Ala-NH₂,

6. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(Bz)₂]-Leu-Arg-Pro-D-Ala-NH₂,

7. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(Car)₂]-Leu-Arg-Pro-D-Ala-NH₂,

8. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂,

9. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(EtCar)₂]-Leu-Arg-Pro-D-Ala-NH₂,

10. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(For)₂]-Leu-Arg-Pro-D-Ala-NH₂,

11. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(PRL)₂]-Leu-Arg-Pro-D-Ala-NH₂,

12. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(CHC)₂]-Leu-Arg-Pro-D-Ala-NH₂,

13. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(Bz)₂]-Leu-Arg-Pro-D-Ala-NH₂,

14. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂,

15. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(For)₂]-Leu-Arg-Pro-D-Ala-NH₂,

16. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(Car)₂]-Leu-Arg-Pro-D-Ala-NH₂,

17. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(EtCar)₂]-Leu-Arg-Pro-D-Ala-NH₂,

18. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(PRL)₂]-Leu-Arg-Pro-D-Ala-NH₂,

19. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(LAU)₂]-Leu-Arg-Pro-D-Ala-NH₂,

20. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(Bz)₂]-Leu-Arg-Pro-D-Ala-NH₂,

21. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(CHC)₂]-Leu-Arg-Pro-D-Ala-NH₂,

22. Ac-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys[A₂ pr(Car)₂]-Leu-Arg-Pro-D-Ala-NH₂,

23. Ac-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys[A₂ pr(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂,

24. Ac-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys[A₂ pr(For)₂]-Leu-Arg-Pro-D-Ala-NH₂,

25. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂pr]-Leu-Arg-Pro-D-Ala-NH₂,

26. Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂bu]-Leu-Arg-Pro-D-Ala-NH₂.

The LHRH antagonizing properties of the compounds of this invention makethe compounds useful in human and veterinary practice. For instance, thecompounds of Formula I find use as agents for revealing thecomplications from the undesirable physiological availability ofpituitary gonadotropins in a mammal. Such complications includeprecocious puberty; hormone dependent tumors such as malignant andbenign prostate tumors, e.g. secondary amenorrhea; endometriosis andovarian and mammary cystic diseases in both animals and humans. Thecompounds of Formula I are also useful for regulating ovulation, thusrendering them useful agents for controlling fertility, e.g. asprecoital or postcoital contraceptives, for synchronizing estrus inlivestock and for improving the "rhythm" method. Also, the compounds areuseful for regulating the human menopausal gonadotropin,follicle-stimulating hormone (FSH) and luteinizing hormone (LH) duringperimenopausal and postmenopausal periods in women. As they suppress thespermatogenesis and testosterone level in male they may be potential usefor male contraception.

The peptides of the invention are often administered in the form ofpharmaceutically acceptable, nontoxic salts, such as acid additionsalts. Illustrative of such acid addition salts are hydrochloride,hydrobromide, sulphate, phosphate, fumarate, gluconate, tannate,maleate, acetate, citrate, benzoate, succinate, alginate, pamoate,malate, ascorbate, tartrate, and the like.

Microcapsules or microparticles of these peptides formulated frompoly(DL-lactide-co-glycolide) may be the preferred sustained deliverysystems. Intravenous administration in isotonic saline, phosphate buffersolutions or the like may be also used.

The pharmaceutical compositions will usually contain the peptide inconjunction with a conventional, pharmaceutically-acceptable carrier.Usually, the dosage, will be from about 1 to 100 micrograms of thepeptide per kilogram of the body weight of the host when givenintravenously. Overall, treatment of subjects with these peptides isgenerally carried out in the same manner as the clinical treatment usingother agonists and antagonists of LHRH.

These peptides can be administered to mammals intravenously,subcutaneously, intramuscularly, intranasally to achieve LHRHantagonizing and antitumor effect. Effective dosages will vary with theform of administration and the particular species of mammal beingtreated. An example of one typical dosage form is a physiological salinesolution containing the peptide which solution is administered toprovide a daily dose in the range of about 0.01 to 0.05 mg/kg of bodyweight.

Although the invention has been described with regard to its preferredembodiments, it should be understood that changes and modificationsobvious to one having the ordinary skill in this art may be made withoutdeparting from the scope of the invention, which is set forth in theclaims which are appended thereto. Substitutions known in the art whichdo not significantly detract from its effectiveness may be employed inthe invention.

ASSAY PROCEDURES

The compounds of this invention exhibit powerful effect on gonadotropinrelease by the pituitary, bind to tumor cell membranes and inhibit [³H]thymidine incorporation into DNA in cell cultures.

(a) LH-RH-inhibiting activities

Ability of compounds to influence LH release in vitro is assayed byusing a superfused rat pituitary cell system [S. Vigh and A. V. Schally,Peptides, 5 Suppl. 1, 241-247 (1984); V. Csernus and A. V. Schally, inNeuroendocrine Research Methods, Ed. B. Greenstein, Harwood AcademicPublishers, London, (1990)].

LHRH inhibiting effect of peptides is assayed as follows: each peptideis perfused through the cells for 9 min (3 ml perfusate) at 1 nM.Immediately after that, a mixture containing the same concentration ofpeptide and 3 nM LHRH is administered for 3 min. This was followed byfour consecutive infusions of 3 nM LHRH for 3 min (1 ml perfusate) at 30min intervals (30, 60, 90, 120 min). LH content of the 1 ml fractionscollected is determined by radioimmunoassay (RIA).

(b) In vivo antiovulatory activity of peptides is determined in4-day-cycling rats as described [A. Corbin and C. W. Beattie, Endocr.Res. Commun., 2, 1-23 (1975)].

(c) Receptor binding

Affinity of peptides to rat pituitary and human breast cancer cellmembranes is determined by using labelled LHRH and [D-Trp⁶ ]LHRH. Theassay is carried out similarly to that described by T. Kadar et al.,Proc. Natl. Acad. Sci. USA, 85, 890-894 (1988) and M. Fekete et al.,Endocrinology, 124, 946-955 (1989).

(d) In vivo effect on LH and FSH levels was measured as described by L.Bokser et al. (Proc. Natl. Acad. Sci. US, accepted for publication.)Castrated male rats weighing 350-410 grams anaesthetized with urethanewere injected subcutaneously with Peptide 7 and 8 in doses of 1.25 μgand 5.0 μg. Blood samples were taken from the jugular vein beforeinjection and 1, 2, 3, 4, 6, 24 and 48 hours after the administration ofpeptides. Control animals were injected only with saline. LH and FSHlevels were determined by specific RIAs.

(e) Cytotoxicity test

Ability of peptides of Formula I to inhibit incorporation of [³H]thymidine into DNA of monolayer cultures the human mammary tumor cellline MCF-7 is assayed as described [V. K. Sondak et al., CancerResearch, 44, 1725-1728 (1984); F. Holzel et al., J. Cancer Res. Clin.Oncol. 109, 217-226 (1985); M. Albert et al., J. Cancer Res. Clin.Oncol. 109, 210-216 (1985)].

(f) In vivo antitumor effect

Inhibition of growth of cancerous tumors in rats with compounds ofFormula I was tested as described by Szende et al. (J. Natl. CancerInst., 82, 513-517, 1990; Szende et al., Cancer Research), 50,3716-3721, 1990) by A. V. Schally and T. Redding (Proc. Natl. Acad. Sci.US, 84, 7279-7282, 1987), and by E. Korkut et al. (Proc. Natl. Acad.Sci. US, accepted for publication). Peptide 8 was dissolved in 45%aqueous propylene-glycol and was administered at a dose of 25 μg/dayfrom an ALZET minipump to male rats bearing the androgen-dependentwell-differentiated R3327 Dunning rat prostate adenocarcinoma. Tumorswere measured weekly with microcalipers and tumor volumes werecalculated. Duration of the treatment was 8 weeks, changing theminipumps at the end of the 4th week.

SYNTHESIS OF PEPTIDES

The peptides of the present invention may be prepared by any techniquesthat are known to those skilled in the peptide art. A summary of thetechniques so available may be found in M. Bodanszky, Principles ofPeptide Synthesis, Springer-Verlag, Heildelberg, 1984. Classicalsolution synthesis is described in detail in the treatise "Methoden derOrganische Chemie" (Houben-Weyl), Vol. 15, Synthese von Peptiden, PartsI and II, Georg Thieme Verlag, Stuttgart, 1974. The techniques ofexclusively solid-phase synthesis are set forth in the textbook of J. M.Stewart and J. D. Young, Solid Phase Peptide Synthesis, Pierce Chem Co.,Rockford, Ill., 1984 (2nd ed.) and in the review of G. Barany, et al.,Int. J. Peptide Protein Res. 30, 705-739, 1987.

The basic peptides of this invention were synthesized by solid-phasemethod, but in some cases the side chain at position 6 were built in by"classical" procedure. In the solid phase synthesis, suitable protectedamino acids (sometimes protected peptides) are added stepwise in C→Ndirection once the C-terminal amino acid has been appropriately attached(anchored) to an inert solid support (resin). After completion of acoupling step, the N-terminal protecting group is removed from thisnewly added amino acid residue and the next amino acid (suitablyprotected) is then added, and so forth. After all the desired aminoacids have been linked in the proper sequence, the peptide is cleavedfrom the support and freed from the remaining protecting group(s) undercondition that are minimally destructive towards residues in thesequence. This must be followed by a prudent purification and scrupulouscharacterization of the synthetic product, so as to ensure that thedesired structure is indeed the one obtained.

PREFERRED EMBODIMENT OF SYNTHESIS

A particularly preferred method of preparing compounds of Formula I inthe present invention is solid phase synthesis, but they can also besynthesized by combining the solid phase and classical (solution)methods. In this particularly preferred method, the α-amino function ofthe amino acids is protected by an acid or base sensitive group. Suchprotecting groups should have the properties of being stable to theconditions of peptide linkage formation, while being readily removablewithout destruction of the growing peptide chain or racemization of anyof the chiral centers contained herein.

The peptides of Formula I are preferably prepared from intermediatepeptides of Formula IV:

    X.sup.1 --R.sup.1 --D-Phe(4HI)-R.sup.3 -Ser(X.sup.4)-R.sup.5 (X.sup.5)--R.sup.6 (X.sup.6)-Leu-Arg(X.sup.8)-Pro-D-Ala-NH--X.sup.10IV

wherein

R¹, R³, R⁵, R⁶, HI and X¹ are as defined hereinabove,

X⁴ is a protecting group for the hydroxyl group of serine, such asbenzyl (Bzl) or 2,6-dichlorobenzyl (DCB). The preferred protecting groupis Bzl.

X⁵ is benzyl, 2-Br-benzyloxycarbonyl or DCB (preferred) to protect thephenolic hydroxyl of R⁵ Tyr;

is Tos (preferred), nitro or methyl-(t-butylbenzene)-sulfonyl to protectthe guanidino group if R⁵ is Arg,

X⁶ is a protecting group for side chain amino group of Lys or Orn, suchas Z, Z(2-CI) (preferred) or FMOC,

X⁸ is a protecting group for the Arg and may be nitro,methyl-(t-butylbenzene)sulfonyl or Tos (preferred).

X¹⁰ is an amide to protect the benzhydryl or methylbenzhydryl groupincorporated into resin support; for synthesis of peptide amides, thecommercially available benzhydrylamino-polystyrene-2% divinylbenzenecopolymer is preferred.

The solid phase synthesis of the peptides of Formula IV is commenced bythe attachment of Boc-protected D-Ala to a benzhydrylamine resin in CH₂Cl₂. The coupling is carried out using DIC or DIC/HOBt at ambienttemperature. After the removal of the Boc group, the coupling ofsuccessive protected amino acids (each is applied in a 3 molar excess)is carried out in CH₂ Cl₂ or in mixtures of DMF/CH₂ Cl₂ depending on thesolubility of Boc-amino acids. The success of coupling reaction at eachstage of the synthesis is preferably monitored by the ninhydrin test asdescribed by Kaiser et al. [Anal. Biochem. 34, 595 (1970)]. In caseswhere incomplete coupling occurs, the coupling procedure is repeatedbefore removal of the alpha-amino protecting group prior to the reactionwith the next amino acid.

After the desired amino acid sequence of intermediate peptides ofFormula IV has been completed, the N-terminal acetylation is carried outusing Ac₂ O/TEA, and the peptide-resin is then treated with liquid HF inthe presence of anisole to yield the peptides of Formula IV wherein X⁴,X⁵, X⁶, X⁸, and X¹⁰ are hydrogens.

These peptides are converted into peptides of Formula I (wherein Y isY¹) by carbodiimide coupling method with preformed2,3-bis-benzoyl-diaminopropionic acid,2,3-bis-cyclohexanoyl-diaminopropionic acid,2,3-bis-lauroyl-diaminopropinoic acid, 2,4-bis-benzoyl-diaminobutyricacid, 2,4-bis-cyclohexanoyl-diaminobutyric acid,2,4-bis-lauroyl-diaminobutyric acid.

To produce compounds of Formula I, wherein Y¹ is lower alkanoyl and Y²is lower carbamoyl, the second synthetic method is preferred because ofthe high hydrophilicity of the substituents on the lysine⁶ side chain,i.e. for example 2,3-bis-formyl-diaminopropionic acid. These kinds ofpeptides are prepared from compounds of Formula V:

    X.sup.1 --R.sup.1 --D-Phe(4HI)--R.sup.3 -Ser(X.sup.4)--R.sup.5 (X.sup.5)--R.sup.6 [A(X.sup.6').sub.2 ]-Leu-Arg(X.sup.8)-Pro-D-Ala-NH--X.sup.10                 VI

wherein

R¹, R³, R⁵, R⁶, HI and X¹ are as defined hereinabove.

X⁴ is a protecting group for the hydroxyl group of serine, such asbenzyl (Bzl) or 2,6-dichlorobenzyl (DCB). The preferred protecting groupis Bzl.

X⁵ is benzyl, 2-Br-benzyloxycarbonyl or DCB (preferred) for protectingthe phenolic hydroxyl of R⁵ Tyr; or

is Tos (preferred), nitro or methyl-(t-butylbenzene)-sulfonyl to protectthe guanidino group if R⁵ is Arg,

X^(6') is an amino protecting group for the diaminoacyl side chain ofLys, such as Z, Z(2-Cl) or FMOC,

X⁸ is suitable for protecting the Arg group; such as nitro,methyl-(t-butylbenzene)-sulfonyl or Tos (preferred),

X¹⁰ is an amide protecting benzhydryl or methylbenzhydryl groupincorporated into resin support; for synthesis of peptide amides, thecommercially available benzhydrylamino-polystyrene-2% divinylbenzenecopolymer is preferred.

Preparation of all protected intermediate peptides of Formula V iscarried out by solid phase peptide synthesis, as described for peptideshaving the Formula IV, but a suitably protected R⁶ (A) residue,preferably Boc-R⁶ [A(FMOC)₂ ], is incorporated in position 6 instead ofBoc-R⁶ X⁶. The protecting group on A is chosen to be selectivelyremovable, while the other protecting group stay intact during theremoval of the two X^(6'). This step can be solved for example by thecleaving FMOC blocking groups with piperidine supplying peptides ofFormula Va on resin:

    X.sup.1 --R.sup.1 --R.sup.2 --R.sup.3 -Ser(X.sup.4)-R.sup.5 (X.sup.5)--R.sup.6 (A)-Leu-Arg(X.sup.8)-Pro-D-Ala-NH--X.sup.10Va

wherein X¹, R¹, R², R³, R⁵, R⁶ and A are as defined hereinabove, and X⁴,X⁵, X⁶ and X¹⁰ are not hydrogen.

The free amino groups at position 6 are then acylated with formicacid-Ac₂ O mixture, or with halides or anhydride of acetic acid,propionic acid or pivalic acid to give compounds of Formula I, wherein Yis Y¹ after deprotection.

Splitting off the protecting groups and cleavage of the peptides fromthe resin occurred after the formation of the side chain on R⁶.

In an alternate synthesis, fully deprotected peptides of Formula Vb areobtained by deprotection of intermediate peptides of Formula V in whichpreferably Boc-R⁶ [A(Z)₂ ], incorporated in position 6 instead of Boc-R⁶[A(FMOC)₂ ]:

    X.sup.1 --R.sup.1 --R.sup.2 --R.sup.3 -Ser-R.sup.5 --R.sup.6 (A)-Leu-Arg-Pro-D-Ala-NH.sub.2                            Vb

wherein X¹, R¹, R², R³, R⁵, R⁶ and A are as defined hereinabove.

The process for producing peptides of Formula I with A(Y²)₂ side chaincomprises reacting a peptide of Formula Vb with a source of suitablecyanate, suitably metal-cyanates, e.g. potassium cyanate or an N-alkylisocyanate, e.g. N-ethyl-isocyanate.

An easy way to produce compounds of Formula I wherein Y is Y¹ is thedirect acylation of the diamino residue at position 6 of peptides ofFormula Vb with equivalent amount of acyl-halide, with AcO₂ /HCOOHmixture, with acetyl- or propionyl-imidazyl. The reactions arestraightforward giving single compounds despite of the presence of freeOH group on serine⁴.

An alternative synthetic method for preparing peptides of Formula I isincorporating the suitable protected bis-substituted-diaminoacyl-R⁶instead of protected R⁶. The synthesis is carried out exactly asmentioned above except that Boc-R⁶ (AY₂) is incorporated into thepeptide instead of Boc-R⁶ (X⁶) at the fifth step of the synthesis.

Purification of Peptides

Crude synthetic products (>500 mg) were purified on a BECKMAN Prep-350preparative HPLC system equipped with a DYNAMAX MACRO column (41.4×250mm) packed with spherical C18 silica gel (pore size: 300 Å, particlesize: 12 μm) (RAININ Inc., Co., Woburn, Mass.) (Column A). Purificationof smaller amount of peptides (<250 mg) were performed on a BECKMAN HPLCsystem (Model 142) using a DYNAMAX MACRO (21.2×250 mm) column packedwith the same medium, as above (Column B). To purify peptides weighing<50 mg, a reversed phase, 10×250 mm VYDAC Protein & Peptide C₁ 8 column(pore size: 300 Å, particle size: 5 μm) (ALTECH, Deerfield, Ill.)(Column C) or a 10×250 mm W-POREX C₁ 8 column (pore size: 300 Å,particle size: μm) (Phenomenex, Rancho Palos Verdes, Calif.) (Column D)were used. Columns were eluted with solvent system i consisting of (A)0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous acetonitrile usually ina gradient mode. Column eluant was monitored with UV detectors operatingat 230 or 280 nm. Chromatography was effected at ambient temperature.

Analytical HPLC

Analysis of crude and purified peptides was carried out with aHewlett-Packard Model 1090 liquid chromatograph equipped with a diodearray detector set for 220 and 280 nm and a reversed phase 4.6×250 mmW-POREX C₁ 8 column (pore size: 300 Å, particle size: 5 μm) (Column E).A flow rate of 1.2 ml/min of solvent system i or solvent system iiconsisting of (A) 0.05M ammoniumacetate pH=7.0 and (B) 0.05Mammoniumacetate in 65% aqueous acetonitrile was maintained and theseparations were performed at room temperature.

Amino Acid Analysis

Peptide samples are hydrolized at 110° C. for 20 hr in evacuated sealedtubes containing 4M methane-sulfonic acid. Analysis are performed with aBeckman 6300 amino acid analyzer.

PREPARATION I

    bis-benzoyl-2,4-diaminopropionic acid                      Ia

    bis-benzoyl-2,4-DL-diaminobutyric acid                     Ib

To the solution of 140 mg (1 mmol) DL-A₂ pr in 2 ml 10% NaOH, 1.5 ml of25% benzoylchloride in dioxane was added in dropwise at 4° C. Thereaction mixture was mixed for 24 hours at 4° C. then the title compoundwas extracted with ethylacetate and purified by recrystallization fromchloroform-hexane. (Bz)₂ -DL-A₂ bu was prepared similarly but usingDL-A₂ bu instead of A₂ pr. Retention factors are 0.60 and 0.69,respectively, on silicagel TLC plate with solvent systemethylacetate-pyridine-acetic acid-water 60-20-6-11.

PREPARATION II

    bis-cyclohexanoyl-2,3-diaminopropionic acid                IIa

    bis-cyclohexanoyl-2,3-DL-diaminobutyric acid               IIb

140 mg (1 mmol) DL-A₂ pr.HCl in 2 ml of 10% NaOH was stirred for 24hours at room temperature with 1.6 ml (3 mmol) 25% cyclohexanecarbonylchloride in dioxane added by dropwise. The title compound was purifiedby solvent extraction and recrystallized from benzene-hexane.

Preparation IIb was made in a similar manner except using 191 mg (1mmol) DL-2,4-diaminobutyric acid.2HCl instead of 2,3-diaminopropionicacid. The retention factors are 0.69 and 0.79, when chromatographed onsilicagel TLC in solvent system as described in preparation I.

PREPARATION III

    Boc-D-Lys(A.sub.2 pr)-OH                                   IIIa

    Boc-D-Lys(DL-A.sub.2 bu)-OH                                IIIb

To a DMF solution (4 ml) of a mixed anhydride, prepared from Z₂ -A₂ pr(0.72 g) and ethyl-chloroformate (0.2 ml) in the presence of TEA (0.28ml), 4 ml DMF containing 0.5 g N.sup.α -Boc-D-Lys and 0.3 ml TEA wereadded with stirring at 0° C. After two hours, the reaction mixture wasconcentrated to an oil under reduced pressure, dissolved in water andethylacetate and acidified with 1M KHSO₄. The organic phase was washedwith water, dried over Na₂ SO₄ and evaporated under vacuum. 0.5 g ofthis protected dipeptide was dissolved in 25 ml 50% aqueous acetic acidand hydrogenated at room temperature for 2 hours in the presence of 0.1g Pd/C (10%). The reaction mixture was filtered and evaporated todryness. The resulting white product was rubbed with diethylether,filtered and dried.

Preparation IIIb was prepared in a similar manner but acylating with Z₂-DL-A₂ bu instead of Z₂ -A₂ pr.

PREPARATION IV

    (FMOC).sub.2 A.sub.2 pr-OH

0.5 g (3.55 mmol) 2,3-diaminopropionic acid was dissolved in 7.1 ml NNaOH and 2.75 g (15% excess) FMOC-OSu in 25 ml acetone was addeddropwise while stirring the mixture at room temperature. After 4 hoursstirring, 3.55 ml N H₂ SO₄ was added, the reaction mixture was filtered,washed with 3×10 ml water and air-dried on the funnel. The product wasrecrystallized from ethylacetate-petroleum ether. The purity of thewhite precipitate (weighted 1.9 g) was checked on silica gel TLC withthe following solvent system: ethylacetate-pyridine-aceticacid-water=120:20:6:11 (R_(f) =0.62-0.66).

PREPARATION V

    Boc-D-Lys[(FMOC).sub.2 A.sub.2 pr]--OH

0.73 g (3 mmol) N.sup.α -Boc-D-Lys-OH was suspended with 0.42 ml (3mmol) TEA in 3 ml 50% aqueous DMF. Then 1.8 g (3.2 mmol) (FMOC)₂ A₂ pr(Preparation VIII) and 0.37 g HOBt was dissolved in 3 ml DMF and wasmixed with 0.5 ml DIC at 0° C. After 10 min this solution was added tothe Boc-D-Lys-OH suspension. On stirring at room temperature, thereaction mixture became clear in 1 hour. Pouring the mixture into 30 mlwater, yielded a yellowish voluminous precipitate which crystallized byrubbing with diethyl ether and recrystallized from MeOH-DCM-hexanesolution (1.2 g). The title compound proved to be homogeneous onsilicagel TLC (R_(f) =0.68-0.72) developed with solvent systemethylacetate-pyridine-acetic acid-water 960:20:6:11.

PREPARATION VI

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH.sub.2VIa

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys-Leu-Arg-Pro-D-Ala-NH.sub.2VIb

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys-Leu-Arg-Pro-D-Ala-NH.sub.2VIc

Boc-D-Ala was attached to 1 g (about 1 mmol) neutralized benzhydrylamineresin containing 1 meq NH₂ (Advanced Chemtech, Louisville, Ky.) by meansof N,N'-diisopropylcarbodiimide (DIC)/1-hydroxybenztriazole (HOBt)mediated coupling for about 2 hours at room temperature indichloromethane or DMF. The coupling of successive protected amino acidswere carried out in a reaction vessel for manual solid phase synthesisusing 2.5-3.0 molar excess of protected amino acids in accordance withthe scedule as follows:

    ______________________________________                                                                        MIXING                                                                        TIMES                                         STEP  REAGENTS AND OPERATIONS   (Min)                                         ______________________________________                                        1     Coupling: Boc-amino acid in                                                                             60-90                                               DCM or DMF depending on the solubility of                                     the particular protected amino acid, plus DIC                           2     iPrOH (or DME then iPOH) wash                                                                           2                                             3     DCM wash                  2                                             4     iPrOH wash                2                                             5     DCM wash (three times)    2                                             6     Deprotection: 50% TFA in DCM twice)                                                                     5 and 25                                      7     DCM wash                  2                                             8     iPrOH wash                1                                             9     Neutralization: 10% TEA in DCM                                                                          2                                             10    iPrOH wash                1                                             11    Neutralization: 10% TEA in DCM                                                                          2                                             12    iPrOH wash                1                                             13    DCM wash (three times)    2                                             ______________________________________                                    

After attaching Boc-Ala to the resin, the following amino acids werethen coupled successively by the same cycle of events: Boc-Pro,Boc-Arg(Tos), Boc-Leu, Boc-D-Lys[Z(2-Cl)], Boc-Tyr(Bzl), Boc-Ser(Bzl),Boc-D-Pal(3), Boc-D-Phe(4Cl), and Boc-D-Nal(2).

Using Boc-Arg(Tos) instead of Boc-Tyr(Bzl) leads to the peptide resinhaving the structure ofBoc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser(Bzl)-Arg(Tos)-D-Lys[Zl-(2-Cl)]-Leu-Arg(Tos)-Pro-D-Ala-NH-RESIN.Likewise, changing Boc-D-Pal(3) to D-Trp in position 3 resulted in thepeptide-resinBoc-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser(Bzl)-Arg(Tos)-D-Lys[Zl-(2Cl)]-Leu-Arg(Tos)-Pro-D-Ala-NH-RESIN.

The decapeptide-resin (3-3.5 g) containing free N-terminal amino groupwas treated with 50-fold excess acetic anhydride and TEA in 30 ml of DMFfor 30 min. The acetylated peptide-resin then was washed with DMF (3times), iPrOH (3 times) and DCM (3 times) and dried in vacuo. Removal ofthe protecting groups and cleavage of the decapeptide from the resin wascarried out by treatment of 1.5-2 g of material with liquid HF (30 ml),anisole (3 ml) at 0 C. for 45 min. The hydrogen fluoride was eliminatedunder a stream of nitrogen and the peptide was precipitated by additionof diethylether. The peptide was then extracted with 50% aqueous aceticacid (3 times), separated from the resin by filtration, diluted withwater and lyophilized.

Crude peptides were purified on Column A with solvent system i using alinear gradient of 40-70% B in 60 min for Preparation Vla and 20-60% Bin 80 min for Preparation Vlb and Vlc. HPLC retention times ofPreparation Vla (837 mg), Vlb (540 mg) and Vlc (521 mg) are 25.5 min,11.4 min and 18.8 min, respectively, when using solvent system i inlinear gradient mode (30-60% B in 30 min). Amino acid analysis gave theexpected results.

PREPARATION VII

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser(Bzl)-Tyr(Bzl)-D-Lys(A.sub.2 pr)-Leu-Arg(Tos)-Pro-D-Ala-NH-resin                       VIIa

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser(Bzl)-Arg(Tos)-D-Lys(A.sub.2 pr)-Leu-Arg(Tos)-Pro-D-Ala-NH-resin                       VIIb

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser(Bzl)-Arg(Tos)-D-Lys(A.sub.2 pr)-Leu-Arg(Tos)-Pro-D-Ala-NH-resin                       VIIc

Preparation of VIIa is carried out by solid phase peptide synthesis inaccordance with the procedures set forth in the schedule of PreparationVI. The decapeptide is built up in ten successive steps couplingBoc-D-Ala to 1 g benzhydrylamine resin first, followed by Boc-Pro,Boc-Arg(Tos), Boc-Leu, Boc-D-Lys[A₂ pr(FMOC)], Boc-Tyr(Bzl),Boc-Ser(Bzl)Boc-D-Pal(3), Boc-D-Phe(4Cl) and Boc-D-Nal(2). N-Terminalacetylation is performed with a 50-fold excess of acetic anhydride inDMF for 30 min. FMOC protecting groups on A₂ pr were removed by treatingthe peptide resin with 20 ml 50% piperidine in DMF for 18 h and thewashed with DMF (3 times), iPrOH (3 times) and DCM (3 times) and kept ina desiccator till the next reaction.

Proceeding in a similar manner but incorporating Boc-Arg(Tos) in placeof Boc-Tyr(Bzl) at position 5,Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser(Bzl)-Arg(Tos)-D-Lys(A₂pr)-Leu-Arg(Tos)-Pro-D-Ala-NH-resin(Preparation VIIb) is prepared. UsingBoc-D-Trp instead of Boc-D-Pal(3) at position 3 and Boc-Arg(Tos) insteadof Boc-Tyr(Bzl) at position 5 results inAc-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser(Bzl)-Arg(Tos)-D-Lys(A₂pr)-Leu-Arg(Tos)-Pro-D-Ala-NH-resin (Preparation VIIc).

PREPARATION VIII

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys(A.sub.2 pr)-Leu-Arg-Pro-D-Ala-NH.sub.2                            VIIIa

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys(A.sub.2 pr)-Leu-Arg-Pro-D-Ala-NH.sub.2                            VIIIb

    Ac-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys(A.sub.2 pr)-Leu-Arg-Pro-D-Ala-NH.sub.2                            VIIIc

The peptides of VIIIa, VIIIb and VIIIc were prepared by the solid-phasetechnique on benzhydrylamine HCl resin in accordance with the proceduresset forth in the Schedule of Preparation VI.

Thus, the resin (0.5 g containing about 0.5 mmole NH₂) is treated duringthe ten successive coupling cycles with Boc-D-Ala, Boc-Pro,Boc-Arg(Tos), Boc-Leu, Boc-Lys[A₂ pr(Z)₂ ], Boc-Tyr(Bzl), Boc-Ser(Bzl),Boc-D-Pal(3), Boc-D-Phe(4Cl), Boc-D-Nal(2) and finally with Ac₂O/imidazole to yield a peptide-resin which is then treated with HF andanisole to afford the free, D-Lys(A₂ pr)-containing peptide of VIIIa.

Proceeding in a similar manner but incorporating Boc-D-Trp in place ofBoc-D-Pal(3) at position 3, the free, D-Lys(A₂ pr)-containing peptide ofVIIIc was prepared (500 mg).

Alternatively, Preparation VIIIa, VIIIb and VIIIc are obtained fromPreparation VIa, VIb and VIc by acylation with Boc₂ -A₂ pr incarbodiimide reaction in the presence of HOBt. Boc groups are thenremoved by treatment with 50% TFA in DCM, the peptide was precipitatedwith diethyl-ether, filtered and dried in vacuo.

Crude peptides were purified on Column A with a gradient of solventsystem i (20-60% B in 80 min). HPLC retention times of VIIIa, VIIIb andVIIIc are 15.1 min, 10.1 min and 17.5 min, respectively, when usingsolvent system i in a linear gradient mode (30-50% B in min).

PREPARATION IX

    Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys(DL-A.sub.2 bu)-Leu-Arg-Pro-D-Ala-NH.sub.2

Preparation IX is prepared by solid phase peptide synthesis as decribedfor Preparation VIIIA with the exception that Boc-D-Lys[DL-A₂ bu(Z)₂ ]is built into the peptide chain in position 6 instead of Boc-D-Lys[A₂pr(Z)₂ ]. HPLC retention time of Preparation IX is 10.4 min when usingsolvent system i in a linear gradient mode (35-50% B in 15 min).

EXAMPLE 1

The peptide Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂ (8) was prepared on solid phase by acetylatingthe free amino groups on A₂ pr substituted Lys side chain of PreparationVII (0.3 g) with 470 μl acetyl-imidazole in the presence of 700 μl TEA.The peptide was then deprotected and split from the resin in one stepusing liquid HF as described for Preparation VI. The crude peptide waspurified by HPLC on Column B eluted with solvent system i using lineargradient (25-50% B in 45 min).

EXAMPLE 2

The syntheses of Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂pr(Bz)₂ -Leu-Arg-Pro-D-Ala-NH₂ (13) andAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(Bz)₂-Leu-Arg-Pro-D-Ala-NH₂ (6) were accomplished by the coupling ofPreparation VIb and 2,3-bis-benzoyl-diaminopropionic acid (PreparationIa) with carbodiimide. A solution (200 μl DMF) of 7 mg Preparation Iaand 3.1 mg HOBt was cooled to 0° C. then reacted with 3.5 μl DIC for 15min. 36.3 mg Preparation VIb dissolved in 200 μl DMF, neutralized withTEA and mixed with the above prepared active ester solution and kept at0° C. for 18 hours. The reaction mixture was directly injected onto theColumn C and purified by eluting with solvent system i to affordcompound 13 (17.6 mg) and 6 (18 mg), respectively.

Following the same procedure, but acylating Preparation VIb and VIc withA₂ pr(AC)₂, Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[(A₂ pr(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂ (2) (19.1 mg) andAc-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys[A₂ pr(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂ (23) (17.2 mg) were prepared.

Acylating Preparation VIb and Via with2,3-bis-cyclohexanoyl-diminopriopionic acid (Preparation IIa) gave 14.8mg Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(CHC)₂]-Leu-Arg-Pro-D-Ala-NH₂ (5) and 15.3 mgAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(CHC)₂]-Leu-Arg-Pro-D-Ala-NH₂ (12). respectively.

Reacting Preparation VIa with 2,4-bis-benzoyl-diaminobutyric acid(Preparation Ib) with 2,4-bis-cyclohexanoyl-diaminobutyric acid(Preparation IIb) or with 2,4-bis-lauroyl-diaminobutyric acid resultedrespectively in peptidesAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(Bz)₂]-Leu-Arg-Pro-D-Ala-NH₂ (20) (16.6 mg),Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(CHC)₂]-Leu-Arg-Pro-D-Ala-NH₂ (21) (14.7 mg) andAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(LAU)₂]-Leu-Arg-Pro-D-Ala-NH₂ (19) (8 mg), respectively.

EXAMPLE 3

The synthesis of Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂bu(Car)₂ ]-Leu-Arg-Pro-D-Ala-NH₂ (16) was carried out by carbamylatingthe two free amino group containing intermediate peptide (PreparationIX). 37 mg Preparation IX was dissolved in 100 μl DMF and the solutionbuffered by addition of 15 μl TEA and 30 μl acetic acid. To thismixture, solution of 48 mg potassium cyanate in 100 μl water was addedand the reaction kept at ambient temperature for 48 hours. The titlepeptide (15.8 mg) was isolated by HPLC purification on Column C usingsolvent system i.

Proceeding in a similar manner but using Preparations VIIIa, VIIIb andVIIIc as precursor, the following peptides were prepared:Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(Car)₂]-Leu-Arg-Pro-D-Ala-NH₂ (7) (12.2 mg),Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(Car)₂]-Leu-Arg-Pro-D-Ala-NH₂ (1) (14.7 mg) andAc-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys[A₂ pr(Car)₂]-Leu-Arg-Pro-D-Ala-NH₂ (22) (11.2 mg).

EXAMPLE 4

Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(PRL)₂]-Leu-Arg-Pro-D-Ala-NH₂ (18) was prepared by propionylation of the twofree amino groups on the substituted Lys side chain of Preparation IX.37 mg intermediate peptide was dissolved in 100 μl DMF, neutralized with7 μl TEA and reacted with 50 μl preformed propionyl-imidazole reagentfor 24 hours at room temperature. The reaction mixture was subjected toHPLC on Column C eluted with solvent system i. Lyophylized fractionscontaining pure peptide yielded 13 mg of the title peptide.

Compounds Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂ (8) (14.1 mg) andAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(Ac)₂]-Leu-Arg-Pro-D-Ala-NH₂ (14) (12.8 mg) were prepared by the methoddescribed in this example but using Preparation VIIIa and Preparation IXas a starting compound, respectively, and acetyl-imidazole as anacylating agent.

EXAMPLE 5

The peptide Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂ pr(PRL)₂]-Leu-Arg-Pro-D-Ala-NH₂ (11) was synthesized by solid phase peptidesynthesis on benzhydrylamine resin (1 g≈1 mmol), as described forPreparation VI. The decapeptide was built up by successive coupling ofthe following protected amino acids (or derivatives): Boc-Ala, Boc-Pro,Boc-Arg(Tos), Boc-Leu, Boc-D-Lys[A₂ pr(PRL)₂ ], Boc-Tyr(Bzl),Boc-Ser(Bzl), Boc-D-Pal(3), Boc-D-Phe(4Cl) and Boc-D-Nal(2). Afteracetylation of the N-terminal amino group, removal of the protectinggroups and cleavage of the decapeptide from the resin were carried outas described for Preparation VI. The crude, lyophylized peptide waspurified on Column C.

EXAMPLE 6

The synthesis of Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂pr(For)₂ ]-Leu-Arg-Pro-D-Ala-NH₂ (3) was carried out by formylation offree amino groups of an intermediate peptide (VIIIa) with preformedmixed anhydride from formic acid and acetic anhydride. To prepare thisanhydride, 960 μl (10 mmole) acetic anhydride was left to react with 390μl (10 mmole) formic acid at 0° C. for 30 min. 37 mg Preparation VIIIawas dissolved in 100 μl DMF, 7 μl TEA and 6.7 μl (50 μmole) of aboveprepared reagent was added and the mixture was kept at 0° C. for 1 hour.Purification of Peptide 3 was achieved by HPLC on Column C eluted withsolvent system i and the yield was 22.8 mg.

Peptides Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Tyr-D-Lys[A₃ pr(For)₂]Leu-Arg-Pro-D-Ala-NH₂ (10),Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(For)₂]-Leu-Arg-Pro-D-Ala-NH₂ (15) andAc-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys[A₂ pr(For)₂]Leu-Arg-Pro-D-Ala-NH₂ (24) were synthesized in the same way with theexception that Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys(A₂pr)-Leu-Arg-Pro-D-Ala-NH₂ (Preparation VIIIa),Ac-D-Nal(2)-D-Phe-(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys(DL-A₂bu)-Leu-Arg-Pro-D-Ala-NH₂ (Preparation IX) andAc-D-Nal(2)-D-Phe(4Cl)-D-Trp-Ser-Arg-D-Lys-A₂ pr)-Leu-Arg-Pro-D-Ala-NH₂(Preparation VIIIc) were used as starting compounds.

EXAMPLE 7

The synthesis of the peptideAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys[A₂ pr(EtCar)₂]-Leu-Arg-Pro-D-Ala-NH₂ was accomplished by reacting intermediatepeptide Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Arg-D-Lys(A₂pr)-Leu-Arg-Pro-D-Ala-NH₂ (Preparation VIIIb) with N-ethylisocyanate. 36mg (20 μmole) at intermediate peptide dissolved in 100 μl DMF, pH wasadjusted with 14 μl (100 μmole) TEA and the peptide was reacted with 3.5μl N-ethylisocyanate at 0° C. for 10 hours. The reaction mixture wasinjected onto Column C and eluted with solvent system i to afford thedesired peptide (21.8 mg).

The syntheses of Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[A₂pr(EtCar)₂ ]-Leu-Arg-Pro-D-Ala-NH₂ (9) andAc-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys[DL-A₂ bu(EtCar)₂]-Leu-Arg-Pro-D-Ala-NH₂ (17) were accomplished by the same manner butusing Ac-D-Nal(2)-D-Phe-(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys(A₂pr)-Leu-Arg-Pro-D-Ala-NH₂ (Preparation VIIIa) and Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys(DL-A₂ bu)-Leu-Ar-g-Pro-D-Ala-NH₂(Preparation IX), respectively.

                  TABLE 1                                                         ______________________________________                                        Preparation and HPLC purification methods                                     for LH-RH antagonist                                                          No. of                                                                              Synthetic Gradient (% B/min) for                                                                         Retention time                               Peptide                                                                             method    purification                                                                             analysis                                                                              (min)                                      ______________________________________                                        1.    3         25-50/40   30-45/15                                                                              12.5                                       2.    2         30-45/45   35-50/15                                                                              10.0                                       3.    6         20-50/50   35-50/15                                                                              8.7                                        4.    7         25-45/50   35-50/15                                                                              11.0                                       5.    2         30-55/50   45-60/15                                                                              12.0                                       6.    2         35-55/60   45-60/15                                                                              9.5                                        7.    3         30-60/60   35-50/15                                                                              8.2                                        8.    1 & 4     25-50/50   35-50/15                                                                              12.5                                       9.    7         25-45/50   40-55/15                                                                              10.6                                       10.   6         30-45/45   35-50/15                                                                              10.3                                       11.   4         30-55/50   35-50/15                                                                              14.8                                       12.   2         25-45/50   55-70/15                                                                              11.9                                       13.   2         25-45/50   35-50/15                                                                              12.3                                       14.   4         25-45/50   35-50/15                                                                              12.8                                       15.   6         25-40/45   35-50/15                                                                              12.3                                       16.   3         35-50/45   35-50/15                                                                              11.8                                       17.   7         25-50/50   35-50/15                                                                              13.9                                       18.   4         30-50/40   35-65/15                                                                              14.7                                       19.   2         50-90/60   80-95/15                                                                              12.0                                       20.   2         35-50/45   45-60/15                                                                              8.0                                        21.   2         50-80/60   50-65/15                                                                              10.9                                       22.   3         30-50/40   45-60/15                                                                              8.0                                        23.   2         35-55/40   45-60/15                                                                              9.3                                        24.   6         35-55/40   40-55/15                                                                              12.0                                       25.   Prep. Villa                                                                             20-50/60   35-50/15                                                                              9.3                                        26.   Prep. IX  20-50/60   35-50/15                                                                              9.1                                        ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Antiovulatory activity and affinity of Ac--D--Nal(2)--D--                     Phe(4Cl)--R.sup.3 --Ser--Arg--D--                                             Lys[A.sub.2 pr(Y).sub.2 ]--Leu--Arg--Pro--D--Ala--NH.sub.2                    peptides for membrane receptors of human breast cancer cells                                         % Blockade of                                                                            Affinity Constant                           No. of         Peptide Ovulation  K.sub.a 1                                                                           K.sub.a 2                             Peptide                                                                             R.sup.3  Y       0.75 μg                                                                          1.5 μg                                                                            nM.sup.-1                                                                           uM.sup.-1                           ______________________________________                                        1     D-Pal(3) Car                  7.07  3.15                                2     D-Pal(3) Ac                   16.35 0.32                                3     D-Pal(3) For                  NB                                        4     D-Pal(3) EtCar                9.71  0.05                                5     D-Pal(3) CHC           40     NB                                        6     D-Pal(3) BZ      10    20     NB                                        22    D-Trp    Car                  4.09  2.67                                23    D-Trp    Ac                   6.87  0.14                                24    D-Trp    For           50     NB                                        ______________________________________                                         *.sup.125 I[DTrp].sup.6 LHRH used as the labelled ligand                      NB, no binding                                                           

                  TABLE 3                                                         ______________________________________                                        Antiovulatory activity and affinity of Ac--D--Nal(2)--D--                     Phe(4Cl)--D--Pal(3)--Ser--Arg--D--                                            Lys[A(Y).sub.2 ]Leu--Arg--Pro--D--Ala--NH.sub.2                               peptides for membrane receptors of human breast cancer cells                                          % Blockade of                                                                           Affinity Constant                           No. of          Peptide Ovulation K.sub.a 1                                                                           K.sub.a 2                             Peptide                                                                             A         Y       0.75 μg                                                                          1.5 μg                                                                           nM.sup.-1                                                                           uM.sup.-1                           ______________________________________                                        7     A.sub.2 pr                                                                              Car                 6.27  5.72                                8     A.sub.2 pr                                                                              Ac                  1.57  6.16                                9     A.sub.2 pr                                                                              EtCar   20    50    30.92 8.57                                10    A.sub.2 pr                                                                              For     67    100   48.29 2.11                                12    A.sub.2 pr                                                                              CHC                 1.68  3.57                                14    DL-A.sub.2 bu                                                                           Ac            20    4.83  0.28                                15    DL-A.sub.2 bu                                                                           For     (25**)                                                                              100   NB                                        16    DL-A.sub.2 bu                                                                           Car           33    NB                                        18    DL-A.sub.2 bu                                                                           PRL     75          21.18 6.17                                19    DL-A.sub.2 bu                                                                           LAU                 0     0                                   21    DL-A.sub.2 bu                                                                           CHC                 NB                                        25    A.sub.2 pr-                   3.49  1.29                                26    DL-A.sub.2 bu-                NB                                        ______________________________________                                         *.sup.125 I[DTrp].sup.6 LHRH used as the labelled ligand                      **dose is 0.375 μg                                                         NB, no binding                                                           

                  TABLE 4                                                         ______________________________________                                        LH-RH inhibiting activities of Ac--D--Nal(2)--D--Phe(4Cl)--                   R.sup.3 --Ser--Arg--D--Lys[A.sub.2 pr(Y).sub.2 ]--                            Leu--Arg--Pro--D--Ala--NH.sub.2                                               antagonists in perfused rat pituitary cell system                             at various molar ratios of antagonist to LH-RH                                % inhibition of LH response at different                                      antagonist to LH-RH ratio                                                     1:1                3:1                                                        No. of                                                                              0       30     60   90   0     30   60   90                             Peptide                                                                             min     min    min  min  min   min  min  min                            ______________________________________                                        1     80      46     31        95    59   55   52                             2     26      27     29   25   88    41   13                                  3                              93    43   26   24                             4                              91    50   42   41                             5                              95    60   60   60                             6     50      40     35   30   82    63   60   55                             22                             64    22   24                                  23                             52    22   21   22                             24    36      11      0    9   58     5   18   27                             ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        LH-RH inhibiting activities of Ac--D--Nal(2)--D--Phe(4Cl)--                   D--Pal(3)--Ser--Arg--D--Lys[A.sub.2 pr(Y).sub.2 ]--                           Leu--Arg--Pro--D--Ala--NH.sub.2                                               antagonists in perfused rat pituitary cell system                             at various molar ratios of antagonist to LH-RH                                % inhibition of LH response at different                                      antagonist to LH-RH ratio                                                     1:1                3:1                                                        No. of                                                                              0       30     60   90   0     30   60   90                             Peptide                                                                             min     min    min  min  min   min  min  min                            ______________________________________                                        7     57      51     45   41   96    78   70   58                             8     64      39     27   22   99    66   47   37                             9                              90    72                                       14                             80    63   51   53                             15    44      41     22   23   99    60   34   21                             16    52      33     24   28                                                  18                             90    71   58   54                             19    20      20      0    0                                                  25    57      49     43        85    69   62   57                             26    70      52     37        90    75   62   57                             ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Effect of 25 μg/day dose of Peptide 8 on the growth                        of Dunning R3327 prostate cancer in rats.                                     Time       Size of tumor (mm.sup.3)                                           (week)     Control group                                                                             Treated group                                          ______________________________________                                        0           4326 ± 1891*                                                                          4001 ± 1617                                         1           6901 ± 2968                                                                           5459 ± 1863                                         2           9174 ± 4507                                                                           5892 ± 1938                                         3           9465 ± 4349                                                                           6357 ± 2327                                         4          12582 ± 6659                                                                            6237 ± 2974**                                      5          12230 ± 4848                                                                            7447 ± 3481**                                      6          14732 ± 6597                                                                            8038 ± 4374**                                      7          17796 ± 8602                                                                             8129 ± 3525***                                    ______________________________________                                         *SD                                                                           **p <0.05                                                                     ***p <0.01                                                               

We claim:
 1. A compound of the formula

    X--R.sup.1 --R.sup.2 --R.sup.3 -Ser-R.sup.5 --R.sup.6 (AY.sub.2)-Leu-Arg-Pro-D-Ala-NH.sub.2

and the pharmaceutically acceptable salts thereof, wherein R¹ is D-Phe,D-Phe(4Cl), D-Nal(1) or D-Nal(2), R² is D-Phe or D-Phe(4Hl), R³ is D-Trpor D-Pal(3), R⁵ is Tyr or Arg, R⁶ is D-Lys or D-Orn, Hl is fluoro,chloro or bromo X is a lower alkanoyl group of 2-5 carbon atoms, A is adiaminoacyl residue having the formula ##STR3## where m is 0 or 1, n is0 or 1, Y is Y¹ or Y², whereinY¹ is an acyl group derived from straightor branched chain aliphatic or alicyclic carboxylic acids having from 3to 12 carbon atoms or aromatic carboxylic acids of 6 or 10 ring carbonatoms, Y² is carbamoyl or a C₁ -C₃ alkyl-substituted carbamoyl grouphaving the formula

    H--(CH.sub.2).sub.n --NH--CO--                             III

where n is 0-3.
 2. A peptide of claim 1, wherein Y is Y¹, where Y¹ isformyl, acetyl, propionyl, butyryl, i-butyryl, cyclohexanoyl or benzoyl.3. A peptide of claim 2, wherein R¹ is D-Nal(2), R² is D-Phe(4Cl), R³ isD-Pal(3), R⁵ is Tyr, R⁶ is D-Lys, X is acetyl and A is2,3-diaminopropionyl.
 4. A peptide of claim 3, wherein Y¹ is formyl. 5.A peptide of claim 3, wherein Y¹ is acetyl.
 6. A peptide of claim 3,wherein Y¹ is propionyl.
 7. A peptide of claim 2, wherein R¹ isD-Nal(2), R² is D-Phe(4Cl), R³ is D-Pal(3), R⁵ Tyr, R⁶ is D-Lys, X isacetyl and A is 2,4-diaminobutyryl.
 8. A peptide of claim 7, wherein Y¹is formyl.
 9. A peptide of claim 7, wherein Y¹ is acetyl.
 10. A peptideof claim 7, wherein Y¹ is propionyl.
 11. A peptide of claim 2, whereinR¹ is D-Nal(2), R² is D-Phe(4Cl), R³ is D-Pal(3), R⁵ is Arg, R⁶ isD-Lys, X is acetyl and A is 2,3-diaminopropionyl.
 12. A peptide of claim11, wherein Y¹ is formyl.
 13. A peptide of claim 11, wherein Y¹ isacetyl.
 14. A peptide of claim 11, wherein Y¹ is propionyl.
 15. Apeptide of claim 2, wherein R¹ is D-Nal(2), R² is D-Phe(4Cl), R³ isD-Trp, R⁵ is Arg, R⁶ is D-Lys, X is acetyl and A is2,3-diaminopropionyl.
 16. A peptide of claim 15, wherein Y¹ is formyl.17. A peptide of claim 15, wherein Y¹ is acetyl.
 18. A peptide of claim1, wherein Y is Y², where Y² is carbamoyl, N-methyl-carbamoyl orN-ethyl-carbamoyl.
 19. A peptide of claim 18, wherein R¹ is D-Nal(2), R²is D-Phe(4Cl), R³ is D-Pal(3), R⁵ is Tyr, or Arg R⁶ is D-Lys, X isacetyl and A is 2,3-diaminopropionyl or 2,4-diaminobutyryl.
 20. Apeptide of claim 19, wherein Y² is carbamoyl.
 21. A peptide of claim 19,wherein Y² is ethylcarbamoyl.